Escherichia coli, as well as all of its gram-negative relatives studied to date, undergo a process of vesiculation, or pinching-off, of the outer membrane. Vesicles have been shown to be capable of fusion with both bacterial and eukaryotic membranes, delivering soluble and membrane components during this process. Although their presence has been recognized for decades, bacterial outer membrane vesicles have yet to be investigated at a basic genetic and biochemical level. In preliminary work, methods have been developed to isolate and purify vesicles from E. coli. The first objective is a genetic approach to elucidate the cellular machinery that produces vesicles. Randomly generated E. coli mutants will be screened for defects in vesicle production. The second objective is to how vesicles benefit the "mother cell" under normal physiological conditions and under stressful growth conditions. The ability of vesicles to communicate between cells may help in a competitive growth environment and to disseminate genetic information. Using biochemical assays, we will investigate vesicle-mediated transmission of proteins, lipids and nucleic acids. Artificial liposomes have been shown to fuse with bacterial membranes in vitro and these experiments provide a basis to begin studying vesicle membrane characteristics that may be important for fusion. Under stressful growth conditions, the vesicle pathway may be co-opted to allow quick remodeling of the outer membrane. The contribution of vesicles to the dramatic switch in lipopolysaccharide composition after cold shock will be analyzed. Further, vesiculation mutants will be used to investigate the function of vesicles during a bacterial response to diverse environmental stresses. The aims of the proposal are distinct and do not depend on one another, yet build on each other to address the project hypotheses. This powerful combination of genetics and biochemistry will set a foundation for future discoveries in this basic area of bacterial physiology. It is anticipated that these studies will reveal general principles of membrane dynamics, while also highlighting new concepts unique to bacterial membranes.